Original compounds useful in cancer therapy are subject of interest in industrial and academic laboratories.
Malignant tumor diseases are the most frequent cause of death. The uncontrolled cellular growth is linked to inherited genetic factors as well as environmental factors. For initiation and development of a malignant disease, the accumulation of several various genetic or epigenetic changes is necessary. This leads to transformation of a healthy cell into a fully malignant phenotype. Cumulation of gene mutations in genes encoding proteins taking part in the regulation of cell division and differentiation, in the control of DNA replication fidelity, in the regulation of apoptosis of the damaged cells, in intercellular communication and intracellular signaling pathways leads to perturbations in normal functioning of these proteins. Malignant cells, unlike benign cells, have the ability to penetrate into the surrounding healthy tissue (invasiveness). Cancer cells can be released from the original tumor and spread through the bloodstream or lymphatic system to distant parts of the body to form new tumors (metastatic process).
The aim of anticancer therapy is to selectively induce apoptosis in the undesirable cancer cells, while not affecting the surrounding healthy tissue. Cytotoxic therapeutics act through DNA damage or microtubule damage and their specificity towards tumor cells in human body is due to their ability to selectively kill fast-proliferating cells. This selectivity can be determined by their cytostatic effects in cell culture in vitro [Chabner B. A., Roberts T. G. (2005), Nat. Rev. Cancer 5, 65-72; Lüllmann H. et al. (2005), Farmakologie a toxikologie, Grada, 15th edition].
The fact that tumor cells are derived from cells of a host organism is a limiting factor for achieving the maximal selectivity of the cytotoxic effect. The sensitivity of cancer cells towards treatment is determined by the growth fraction of a tumor (the ratio of proliferating and non-proliferating tumor cells), the site of action of the cytostatic agent within the cell cycle, and the natural and the acquired resistance of the tumor cells against the cytostatics.
G-quadruplexes are regarded as attractive molecular targets of anticancer therapy of the future [Neidle S. (2011), Therapeutic Applications of Quadruplex Nucleic Acids, Academic Press, 1st edition]. Influencing the stability of DNA G-quadruplexes was identified as one of the regulatory mechanisms for key processes on cellular level. Original compounds useful in influencing the stability of G-quadruplexes are thus of interest for the industry and many academic laboratories. Frequent presence of G-quadruplexes was found in promoter regions of genes, and the physico-chemical and structural characteristics of these DNA structures make them interesting therapeutic targets. Repression of oncogene transcription as a result of stabilization of these four-stranded DNA structures in promoter regions of genes using small molecules is thus one of the pursued strategies of anticancer therapy [Balasubramanian S. et al, (2011), Nature Reviews Drug Discovery 10, 261-275].
Telomeric ends of chromosomes are another area where G-quadruplexes play a key role [Neidle S. (2010), FEBS Journal 277, 1118-1125]. Human telomeres are nucleoprotein complexes containing repeating DNA sequence (5′GGGTTA3′)n (n=100-4000), which has a single strand 24-400 base overhang on its 3′-end [Cimino-Reale G. et al. (2001) Nucleic Acids Res. 29, E35]. Telomeric DNA is gradually getting shorter with each cycle of cell division (so called end replication problem). This process determines the limit of overall number of divisions, which are possible in normal (non-cancer) cells. Majority of cancer cells overcome this end replication problem with help of telomerase mediated extension of telomeric DNA ends. Stabilization of G-quadruplexes in telomeres via small molecules can lead to efficient inhibition of telomerase activity and restoration of the limit for cell division.
Targeting of telomeric G-quadruplexes can influence the function of telomeres also by other means than via inhibition of telomerase. Ends of chromosomes are associated with wide range of proteins, which bind to them. This nucleoprotein complex (so called sheltering complex) is responsible for structural integrity of telomeres in vivo. Small molecules which bind in telomeric region can release proteins from sheltering complex and cause telomere destabilization. This process can lead to apoptosis or replicative senescence. Similar to targeting G-quadruplexes in gene promoters, targeting at telomeric ends of chromosome, which is rich in G-quadruplexes, is also a promising strategy of anticancer therapy.
Currently, small molecules are being sought to induce formation of G-quadruplex-ligand complexes. Such molecules could induce selective inhibition of cancer cell growth [Ou T.-M. et al. (2008), ChemMedChem, 3, 690-713; Phatak P. et al. (2007), Br. J. Cancer, 96, 1223-1233; Di Leva F. S. et al. (2013) J. Med Chem., 56, 9646-54]. Example of such small organic molecule, which is used for G-quadruplex stabilization in vitro and in vivo, is TMPyP4 [Balasubramanian S. et al. (2011), Nature Reviews Drug Discovery 10, 261-275]. A significant drawback is the low selectivity of this particular compound towards G-quadruplex in presence of DNA duplex.
Presently, there are numerous G-quadruplex stabilizers, out of which a few have been tested clinically. In spite of the fact that these compounds limited growth of cancer cells in a promising way, neither has been introduced in clinical practice. For example quarfloxin had poor bioavailability [Quarfloxin (CX-3543); Phase II clinical trials; ClinicalTrials.gov; NCT00780663]. Development of new G-quadruplex stabilizers is a field of research which still receives great attention. Particularly desirable are original small molecules, which are effective in repression of oncogene transcription and, at the same time, show acceptable level of side effects during their applications.
This invention opens a straightforward way for obtaining a brand new class of compounds, which are useful as medicaments for diseases related to increased cellular proliferation and stabilization of G-quaduplexes. These compounds structurally belong to the family of helquats, organic cations based on quaternary nitrogen atom, for which also a non-therapeutic use has been suggested, such as sensitizers for photography [Tani T. (1995) Photographic Sensitivity, Theory and Mechanisms, OUP, Oxford].
Helical extended diquats (helquats) [Adriaenssens et al. (2009), Chem. Eur. J. 15, 1072-1076; Severa et al. (2010), Tetrahedron 66, 3537-3552; Vávra et al. (2012), Eur. J. Org. Chem. 489-499] represent a new and nearly unexplored class of compounds with dicationic helical skeleton. The described basic helquat skeletons are built around two quaternary N-heteroaromatic units, which introduce into this structure two positively charged centers, for example in the form of pyridinium, quinolinium or isoquinolinium cationic units. To this end, the whole arrangement of a typical helquat is associated with dicationicity as well as helical chirality, which is a combination that has not been studied before in the context of small aromatic organic molecules.
Previous efforts focused on helquats allowed only a limited variability of structures which was caused by the fact that each prepared compound was build using multistep synthesis de novo [PV 2009-237]. The follow-up patent application (PV 2013-32, PCT/CZ2014/000009) introduced not only new helquat derivatives but also preparation thereof via one-step diversification of methyl-substituted helquats using Knoevenagel condensation with arylaldehydes.
The present invention describes a completely new class of helquats, which is distinguished by the presence of heteroaryl substituent(s). These compounds are prepared again via one-step diversification of the methyl-substituted helquats using Knoevenagel condensation, but now with substituted or non-substituted heteroarylaldehydes.